CN105424776A - Biosensor based on carbon nano composite material and preparation method thereof - Google Patents
Biosensor based on carbon nano composite material and preparation method thereof Download PDFInfo
- Publication number
- CN105424776A CN105424776A CN201510734068.7A CN201510734068A CN105424776A CN 105424776 A CN105424776 A CN 105424776A CN 201510734068 A CN201510734068 A CN 201510734068A CN 105424776 A CN105424776 A CN 105424776A
- Authority
- CN
- China
- Prior art keywords
- layer
- antibody
- electrode
- graphene oxide
- capture antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Analytical Chemistry (AREA)
- Hematology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Nanotechnology (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Carbon And Carbon Compounds (AREA)
- Hybrid Cells (AREA)
Abstract
The invention discloses a biosensor based on a carbon nano composite material and a preparation method thereof. A platform of the biosensor comprises an electrode, an antibody capture layer attached to the surface of the electrode, a to-be-detected antigen layer and a detection antibody layer. The to-be-detected antigen layer is located between the antibody capture layer and the detection antibody layer. The detection antibody layer is manufactured from a secondary antibody composite. The secondary antibody composite is formed by coupling a detection antibody and the carbon nano composite material. Compared with the prior art, the photoelectric immune biosensor has the advantages that reagents which are used are free of toxin and harm, and a detected life is not corroded or damaged. Meanwhile, the biosensor has the obvious advantages of being high in sensitivity, good in bio-compatibility, capable of displaying detection results more visually and detailedly and the like. Detection instruments are simple, the price is low, and the biosensor can play an important role in the aspect of medical testing and has wide application prospects.
Description
Technical field
The present invention relates to a kind of photoelectricity immunity biosensor based on carbon nano-composite material and preparation method thereof, belong to biosensor technology field.
Background technology
Biology sensor is one of field the most active in current life science and clinical medicine Study on Test Method, has been widely used in the fields such as clinical, industrial, environment and agricultural analysis.Since the nineties in 20th century, the intervention of nanometer technology is that the development of biology sensor provides new vitality, and has achieved breakthrough progress.Nano material is because specific surface area is large, surface reaction activity is high, the not congruent avtive spot increase on surface, catalytic efficiency raising, the adsorptive power of causing of surface atom coordination strengthens, for biological sensing Study of An provides recent studies on approach.Compared with traditional sensor, not only volume is less for novel nano-material sensor, speed is faster but also precision is higher, better reliability.The specific surface area high due to nano particle and the bio-compatibility of itself, play an important role in biological electro catalysis reaction.Such as, enzyme after assembling with nano particle, its activated centre closer to electrode surface, can be easy to carry out electro transfer, improves bioelectrocatalytic activity, makes it more be conducive to applying in electrochemical sensor.Carbon nanomaterial because of the performance of its uniqueness as cheap, wide material sources, be easy to functionalization, excellent electronic property, high strength and wide application prospect and receive much concern.
At present, the photoelectricity probe material being conventionally used to photoelectricity bio-sensing mostly is metallic compound, although this kind of material has excellent opto-electronic conversion performance, but they often have stronger oxidisability or higher toxicity, easily damage is caused to biomolecule or biosome, and under long-term illumination, this compounds easily occurs from corrosion (under illumination, self is unstable).
Optical Electro-Chemistry bioanalysis newly occurs and a kind of analytical approach developed rapidly in recent years, and it is a kind of analytical technology based on Conventional electrochemical.The quantitative basis of this analytical technology depends on the relation between the change of physics between determinand and Optical Electro-Chemistry active substance, photocurrent that chemical interaction produces or photovoltage and the concentration of determinand.Therefore, this method inherits the plurality of advantages that Conventional electrochemical is analyzed, and as cheap, equipment is simple, and sensitivity is high, has again the advantage that some are difficult to realize on Conventional electrochemical platform simultaneously.Such as, the mode that the method adopts light different with electricity two kinds is signal to be excited and detection means, and both can not interfere with each other, and background is low, highly sensitive.
In recent years, carbon nanomaterial, such as carbon nano-tube, Graphene, graphene quantum dot, carbon point etc. are also by a large amount of research for photoelectric analysis bio-sensing field, except not containing except the advantages such as metallic element, wide material sources, good biocompatibility and environmental friendliness, they also show excellent interface regulation and control, electronic conduction and resonance energy transfer ability.By contrast, as the another kind of allotrope of carbon, the research that fullerene-based material (such as C60, C70 etc.) is directly used in photoelectric analysis bio-sensing field is considerably less.
Summary of the invention
Goal of the invention: in order to solve the problems of the technologies described above, the invention provides a kind of photoelectricity immunity biosensor based on carbon nano-composite material and preparation method thereof.
Technical scheme: in order to realize foregoing invention object, the invention discloses a kind of photoelectricity immunity biosensor based on carbon nano-composite material, the platform of described biology sensor comprises with lower part: electrode, the capture antibody layer being close to electrode surface, determined antigen layer and detection antibody layer, and described determined antigen layer is positioned at capture antibody layer and detects in the middle of antibody layer;
Described detection antibody layer is made by two anti-complex, and described two anti-complex are made by detection antibody and the mutual coupling of carbon nano-composite material.
As preferably, described capture antibody layer is made by capture antibody and graphene oxide.
Preferred as another kind, described carbon nano-composite material is the compound substance made by fullerene and graphene oxide.
As preferred further, the weight ratio of described fullerene and graphene oxide is 1:1.
Preferred as another kind, the preparation method of described two anti-complex comprises the following steps:
(1) PTC-NH
2preparation:
Claim to get perylenetetracarboxylic dianhydride, add ethanol, stir, to its dropwise stream add ethylenediamine reagent, continue stirring and spend the night, get precipitation after centrifugal, use ethanol, ultrapure water supersound washing centrifugal respectively, drying at room temperature, obtains PTC-NH
2rear shading is preserved;
(2) C60-GO-PTC-NH
2preparation:
Take Fullerene C20, graphene oxide GO, above-mentioned PTC-NH
2, grind to form fine-powdered, be transferred in PBS, after ultrasonic disperse, stir continuously under room temperature and spend the night, obtain C60-GO-PTC-NH
2;
(3) two anti-complex are prepared:
Get above-mentioned C60-GO-PTC-NH
2, add EDC/NHS, under room temperature, lucifuge stirs, and centrifuge washing, will precipitate resuspended to PBS, in above-mentioned solution, add detection antibody, and 4 DEG C of lucifuges stir, and use PBS centrifuge washing, precipitation be resuspended in PBS, 4 DEG C keep in Dark Place, and obtain described two anti-complex.
Further preferably, the preparation method of described two anti-complex comprises the following steps:
(1) PTC-NH
2preparation:
Take 0.5g perylenetetracarboxylic dianhydride (PTCDA, a kind of water-soluble dye), add 2.5ml ethanol, stir after 1h in 4 DEG C, to its dropwise stream add ethylenediamine reagent 5ml, continue stirring and spend the night, precipitation is got after centrifugal, use ethanol, ultrapure water supersound washing centrifugal twice respectively, drying at room temperature, obtains PTC-NH
2rear shading is preserved.
(2) C60-GO-PTC-NH
2preparation:
Take C60, GO, above-mentioned PTC-NH
2each 0.5mg, grinds to form fine-powdered and is transferred in 1ml0.01MPBS in mortar.After 1min ultrasonic disperse, stir continuously under room temperature and spend the night, obtain C60-GO-PTC-NH
2.
(3) two anti-complex are prepared:
Get C60-GO-PTC-NH
2500ul, adds EDC/NHS500ul (concentration of EDC, NHS is respectively 20mg/ml and 40mg/ml), and under room temperature, lucifuge stirs 1h, and centrifuge washing, will precipitate resuspended to 500ul0.01MPBS.In above-mentioned solution, add 150ul4ug/ml bis-anti-(Ab2 detects antibody), after 4 DEG C of lucifuges stir 5h, use 0.01MPBS centrifuge washing, precipitation be resuspended in 500ul0.01MPBS, 4 DEG C keep in Dark Place, and obtain described two anti-complex.
Preferred as another kind, described electrode is oxidation cigarette tin electrode.
Present invention also offers the preparation method of described photoelectricity immunity biosensor, it is characterized in that, comprise the following steps:
(1) graphene oxide GO is dripped: drip graphene oxide GO at electrode surface, make it be combined in electrode surface completely;
(2) active oxidation Graphene GO: utilize catalyzer, makes the carboxyl on graphene oxide GO activate completely;
(3) covalently bound capture antibody: the graphene oxide GO surface to above-mentioned activation drips capture antibody, and capture antibody is fully connected with graphene oxide GO;
(4) non-specific sites is closed: close the graphene oxide GO site obtained antibody and combine at large, form capture antibody layer;
(5) determined antigen in medium is caught: drip determined antigen to above-mentioned capture antibody layer, fully react, obtain determined antigen layer;
(6) detect antibody layer preparation: wash away above-mentioned unconjugated determined antigen, dry, drip described two anti-complex, carry out lucifuge, room temperature reaction, obtain detection antibody layer, carried out the amount of detectable antigens by the power change of two anti-complex photocurrents.
As preferably, the preparation method of described photoelectricity immunity biosensor, comprises the following steps:
(1) drip graphene oxide GO: get graphene oxide GO, be dispersed in PBS, be added drop-wise to treated oxidation cigarette tin electrode surface, ambient temperatare is put, until it is combined in electrode surface completely;
(2) active oxidation Graphene GO: rinsed in PBS solution by the above-mentioned electrode adding GO and wash, wash away the GO not being combined in electrode surface, drips EDC/NHS, and room temperature is placed, and the carboxyl on GO is activated completely;
(3) covalently bound capture antibody: rinsed in PBS solution by step (2) the electrode obtained and wash, wash away the EDC/NHS do not utilized, dry, drips capture antibody, reacts, capture antibody is fully connected with GO at 4 DEG C;
(4) non-specific sites is closed: rinsed in PBS solution by step (3) the electrode obtained and wash, wash away unconjugated capture antibody, dry, drip bovine serum albumin(BSA) (BSA), room temperature is placed, and closes the site obtained antibody and combine at large, forms capture antibody layer;
(5) catch the determined antigen in medium: rinsed in PBS solution by step (4) the electrode obtained and wash, wash away unnecessary BSA, dry, drip antigen, room temperature reaction, obtains determined antigen layer;
(6) antibody layer preparation is detected: rinsed in PBS solution by step (5) the electrode obtained and wash, wash away unconjugated antigen, dry, drip described two anti-complex, carry out lucifuge, room temperature reaction, obtains detection antibody layer, carrys out detectable antigens amount by the power change of two anti-complex photocurrents.
As preferred further, the preparation method of described photoelectricity immunity biosensor, comprises the following steps:
(1) graphene oxide GO is dripped: get 0.5mg/mlGO (0.01MPBS) 10ul, carefully be added drop-wise to treated oxidation cigarette tin (ITO) electrode surface, ambient temperatare puts 24h, until guarantee that material is combined in electrode surface completely, the primary antibodie after utilizing its rich carboxylic characteristic to make it can combine well and be fixed on electrode;
(2) active oxidation Graphene GO: electrode back having been added GO is rinsed and washed in 0.01MPBS solution, wash away the GO not being combined in electrode surface as far as possible, get EDC/NHS10ul, room temperature places 2h, guarantee that the carboxyl on GO is activated completely, conveniently utilize it in conjunction with follow-up primary antibodie;
(3) covalently bound capture antibody: the electrode of back is rinsed in 0.01MPBS solution and washes, wash away the EDC/NHS do not utilized as far as possible, dry, drip the primary antibodie (Ab1 of 4ug/ml, capture antibody) 10ul, react 12h at 4 DEG C, Ab1 is connected with GO fully, be made into immobilized antibody;
(4) close non-specific sites: rinsed in 0.01MPBS solution by the electrode of back and wash, wash away unconjugated Ab1 as far as possible, dry, drip 1.0wt%BSA10ul.Room temperature places 0.5h, closes not by some sites that Ab1 combines, and prevents these sites from combining with material afterwards, improves background colour, increase error, form capture antibody layer;
(5) catch the determined antigen in medium: rinsed in 0.01MPBS solution by the electrode of back and wash, wash away unnecessary BSA as far as possible, dry, drip the antigen 1 0ul of 1ug/ml, room temperature reaction 2h, obtains determined antigen layer;
(6) antibody layer preparation is detected: rinsed in 0.01MPBS solution by the electrode of back and wash, wash away unconjugated antigen as far as possible, dry, drip the two anti-compound 10ul configured in advance, carry out lucifuge, room temperature reaction 4h, obtains detection antibody layer, carrys out detectable antigens amount by the power change of two anti-complex photocurrents.
So far, the photoelectricity immunity biosensor based on carbon nano-composite material of a layer assembly has built, and its assembling sequence follows following program:
By searching the canonical plotting of antigen concentration and the two anti-compound photocurrent magnitude relationship of drawing by this sensor, this platform can detect the size of different types of Ag concentration in medium by the kind of conversion Ab1, Ab2.
Object of the present invention is intended to, by carbon nanomaterial and the combination such as bio-sensing, life analysis, develop the Photoelectrochemistrbiosensor biosensor based on carbon nanomaterial.By designing and regulating and controlling fullerene molecule surface organo-functional group kind and distribution, improve the dissolubility of fullerene-based material, phase, stability and interfacial assembly behavior; Utilize the electronic transmission performance of graphene oxide composite material excellence and unique bidimensional molecular structure, pass through noncovalent interaction, build fullerene derivate-graphene oxide assembly, research Functional Fullerene and the interfacial assembly of graphene oxide regulate and control and impact on assembly interfacial structure and opto-electronic conversion performance, disclose its interfacial interaction mechanism, develop the novel photoelectric chemo-immunity sensor based on carbon nanomaterial.
In addition, fullerene, carbon nano-tube and Graphene, they are semiconductor material inherently, when being applied to Optical Electro-Chemistry sensing, in the present invention, fullerene can directly as light active material, and do not need other fluorescence molecule of extra introducing as carbon nano-tube and Graphene, thus senser element interface is few, system is relatively simple.
Technique effect: relative to prior art, photoelectricity immunity biosensor of the present invention, agents useful for same is nontoxic, detected life entity is not existed to the advantage of corrosion or other damages; Simultaneously with traditional immunoassay Comparison between detecting methods, have highly sensitive, bio-compatibility is good, more directly perceived, finerly can show the clear superiorities such as testing result, and also effectively can avoid the hidden danger of the ray possibility damage biological molecule existing for radiometric analysis technology.This photoelectricity immune sensing system, detecting instrument is simple, and price is comparatively cheap, can play an important role, be with a wide range of applications in medical science context of detection.
Accompanying drawing explanation
Fig. 1 is C60-PTC-NH
2the photoelectric activity of-GO detects current-vs-time figure;
Fig. 2 is the photoelectricity flow graph before and after the anti-carbon nano-complex of assembling two;
Fig. 3 is the linear relationship chart between antigen concentration and photo-current intensity.
Embodiment
Technical solution of the present invention is further described below in conjunction with accompanying drawing.
Embodiment 1
1, the preparation of two anti-complex:
(1) PTC-NH
2preparation:
Take 0.5g perylenetetracarboxylic dianhydride (PTCDA, a kind of water-soluble dye), add 2.5ml ethanol, stir after 1h in 4 DEG C, to its dropwise stream add ethylenediamine reagent 5ml, continue stirring and spend the night, precipitation is got after centrifugal, use ethanol, ultrapure water supersound washing centrifugal twice respectively, drying at room temperature, obtains PTC-NH
2rear shading is preserved.
(2) C60-GO-PTC-NH
2preparation:
Take C60, GO, above-mentioned PTC-NH
2each 0.5mg, grinds to form fine-powdered and is transferred in 1ml0.01MPBS in mortar.After 1min ultrasonic disperse, stir continuously under room temperature and spend the night, obtain C60-GO-PTC-NH
2.
(3) two anti-complex are prepared:
Get C60-GO-PTC-NH
2500ul, adds EDC/NHS500ul (concentration of EDC, NHS is respectively 20mg/ml and 40mg/ml), and under room temperature, lucifuge stirs 1h, and centrifuge washing, will precipitate resuspended to 500ul0.01MPBS.In above-mentioned solution, add 150ul4ug/ml bis-anti-(Ab2 detects antibody), after 4 DEG C of lucifuges stir 5h, use 0.01MPBS centrifuge washing, precipitation be resuspended in 500ul0.01MPBS, 4 DEG C keep in Dark Place, and obtain described two anti-complex.
2, the preparation of photoelectricity immunity biosensor:
(1) graphene oxide GO is dripped: get 0.5mg/mlGO (0.01MPBS) 10ul, carefully be added drop-wise to treated oxidation cigarette tin (ITO) electrode surface, ambient temperatare puts 24h, until guarantee that material is combined in electrode surface completely, the primary antibodie after utilizing its rich carboxylic characteristic to make it can combine well and be fixed on electrode;
(2) active oxidation Graphene GO: electrode back having been added GO is rinsed and washed in 0.01MPBS solution, wash away the GO not being combined in electrode surface as far as possible, get EDC/NHS10ul, room temperature places 2h, guarantee that the carboxyl on GO is activated completely, conveniently utilize it in conjunction with follow-up primary antibodie;
(3) covalently bound capture antibody: the electrode of back is rinsed in 0.01MPBS solution and washes, wash away the EDC/NHS do not utilized as far as possible, dry, drip the primary antibodie (Ab1 of 4ug/ml, capture antibody) 10ul, react 12h at 4 DEG C, Ab1 is connected with GO fully, be made into immobilized antibody;
(4) close non-specific sites: rinsed in 0.01MPBS solution by the electrode of back and wash, wash away unconjugated Ab1 as far as possible, dry, drip 1.0wt%BSA10ul.Room temperature places 0.5h, closes not by some sites that Ab1 combines, and prevents these sites from combining with material afterwards, improves background colour, increase error, form capture antibody layer;
(5) catch the determined antigen in medium: rinsed in 0.01MPBS solution by the electrode of back and wash, wash away unnecessary BSA as far as possible, dry, drip the antigen 1 0ul of 1ug/ml, room temperature reaction 2h, obtains determined antigen layer;
(6) antibody layer preparation is detected: rinsed in 0.01MPBS solution by the electrode of back and wash, wash away unconjugated antigen as far as possible, dry, drip the two anti-compound 10ul configured in advance, carry out lucifuge, room temperature reaction 4h, obtains detection antibody layer, carrys out detectable antigens amount by the power change of two anti-complex photocurrents.
So far, the photoelectricity immunity biosensor based on carbon nano-composite material of a layer assembly has built, and its assembling sequence follows following program:
By searching the canonical plotting of antigen concentration and the two anti-compound photocurrent magnitude relationship of drawing by this sensor, this platform can detect the size of different types of Ag concentration in medium by the kind of conversion Ab1, Ab2.
First the present invention produces PTC-NH
2, then prepare water-soluble carbon nanomaterial (containing C60) compound by various operation, then by itself and detection antibody (Ab2) coupling; Immobilized good capture antibody in semiconductor ITO electrode, is used for catching the corresponding antigens existed in medium specially simultaneously; The compound with detecting antibody is finally made to be combined with antigentic specificity.Thus reaching under the model of a sandwich double antibody sandwich method, the power of the photocurrent reflected by C60 compound changes the amount indirectly reflecting antigen, obtains the determination of trace of unknown determined antigen.Sensor platform essential structure involved by the method is: capture antibody (immobilized in ITO semiconductor photoelectrode)-determined antigen-detection antibody (coupling C60 compound) three parts; Detect antibody and the mutual coupling of novel carbon nanomaterial, and the energy variation measured in process is electric energy by Change of light energy.
Embodiment 2C60-PTC-NH
2the photoelectric activity of-GO detects
1, instrument: xenon source, Shanghai occasion China's electrochemical workstation (chi660e software)
2, material:
ITO: oxidation cigarette tin semi-conducting electrode;
C60-PTC-NH
2-GO: prepare the carbon nano-complex that coupling two is anti-, 10ul
3, method: by C60-PTC-NH
2-GO10ul drips on ITO, utilizes electrochemical workstation, under the irradiation of xenon lamp (controlled light on/off), measures C60-PTC-NH
2the current versus time curve of-GO/ITO.
4, result:
C60-PTC-NH
2the photoelectric activity of-GO detects current-vs-time relation, sees Figure of description 1.In described Fig. 1, #1 curve is the photo-current intensity of ITO (oxidation cigarette tin semi-conducting electrode); #2 curve represents ITO-C60-PTC-NH
2the photo-current intensity of-GO (semi-conducting electrode has dripped and has prepared the anti-carbon nano-complex of coupling two).The rising of two kinds of curves and drop (i.e. the change of ordinate current value) be due to light give produced with withdrawing.
Bare electrode directly gives light source irradiation and only can excite very little photocurrent (as shown in #2 curve), and after drip synthesized material on electrode, give the photocurrent (as shown in #1 curve) of powerful manyfold before light source irradiation just can excite comparatively instantaneously again.Confirm that synthesized carbon nano-composite material has good photoelectric activity thus, can be used as experiment detection signal probe, itself and two anti-(namely detecting antibody) couplings can be made, the antigen amount captured as immobilized primary antibodie (capture antibody) in sensor platform number an input index.The amount of antigen corresponds to two anti-amounts, and two amounts resisted correspond to the amount with the carbon nano-composite material of two anti-couplings, and the amount of this compound substance can be reflected by the change of photocurrent.In brief, build after whole process completes at sensor platform, the size reflection of photocurrent be exactly the size of antigen concentration in medium.
The fundamental purpose of this experiment is the sensitivity in order to prove the anti-coupled signal pointer with two, and it can produce powerful electric current after running into the exciting of light, and can be used as semiochemicals important in experiment.(qualitative experiment)
The feasibility study of embodiment 3 photoelectricity immune sensing
1, instrument: xenon source, Shanghai occasion China's electrochemical workstation (chi660e software)
2, method: photoelectricity immunity biosensor obtained in embodiment 1 is carried out the test of photoelectricity immune sensing feasibility, namely electrochemical workstation is utilized, under the irradiation of xenon lamp (controlled light on/off), measure the current versus time curve of photoelectricity immunity bio-sensing electrode.
3, result:
Assemble the photocurrent before and after two anti-carbon nano-complexes (i.e. two anti-complex), see Figure of description 2.In described Fig. 2, the photocurrent curve before #2 curve and #1 curve represent two anti-carbon nano-complex assemblings respectively and after assembling.The difference that both photocurrents exist, is two anti-carbon nano-complexes and causes, and then indirectly can draw the amount of antigen contained in medium.(concrete theoretical with above-mentioned Fig. 1)
The fundamental purpose of this experiment is the feasibility in order to investigate photoelectricity immune sensing in the design, namely measures C60-PTC-NH
2the Optical Electro-Chemistry of-GO-Ab2/Ag/Ab1/GO/ITO electrode is active, by semiochemicals C60-PTC-NH
2the signal representation of-GO, the existence of precognition test substance Ag.
Linear relationship between embodiment 4 antigen concentration and photo-current intensity is investigated
By doing the revision test of above-mentioned Fig. 2, if 6 concentration gradients, being respectively 1ug/ml, 0.1ug/ml, 0.01ug/ml, 1ng/ml, 0.1ng/ml, 0.01ng/ml, under each concentration, having done again some parallel controls respectively.
By data processing, between each density antigen drawn and its photocurrent, the canonical plotting of corresponding relation, is shown in Figure of description 3.
Can be drawn by Fig. 3, along with the continuous reduction of antigen concentration, photo-current intensity value also constantly reduces, and linear relationship higher in related coefficient between antigen concentration and photo-current intensity size.
The fundamental purpose of this experiment is the size of the photocurrent corresponding to antigen in order to draw various concentration, and then make by it canonical plotting detected for this kind of antigen meeting this system, with be reached through survey unknown antigen under this sensor systems shown by the size of photocurrent that goes out, and then under typical curve, release the object of Specific amounts of unknown antigen.(quantitatively).
Claims (8)
1. the photoelectricity immunity biosensor based on carbon nano-composite material, it is characterized in that, the platform of described biology sensor comprises with lower part: electrode, the capture antibody layer being close to electrode surface, determined antigen layer and detection antibody layer, and described determined antigen layer is positioned at capture antibody layer and detects in the middle of antibody layer;
Described detection antibody layer is made by two anti-complex, and described two anti-complex are made by detection antibody and the mutual coupling of carbon nano-composite material.
2. photoelectricity immunity biosensor according to claim 1, is characterized in that, described capture antibody layer is made by capture antibody and graphene oxide.
3. photoelectricity immunity biosensor according to claim 1, is characterized in that, described carbon nano-composite material is the compound substance made by fullerene and graphene oxide.
4. photoelectricity immunity biosensor according to claim 3, is characterized in that, the weight ratio of described fullerene and graphene oxide is 1:1.
5. photoelectricity immunity biosensor according to claim 1, is characterized in that, the preparation method of described two anti-complex comprises the following steps:
(1) PTC-NH
2preparation:
Claim to get perylenetetracarboxylic dianhydride, add ethanol, stir, to its dropwise stream add ethylenediamine reagent, continue stirring and spend the night, get precipitation after centrifugal, use ethanol, ultrapure water supersound washing centrifugal respectively, drying at room temperature, obtains PTC-NH
2rear shading is preserved;
(2) C60-GO-PTC-NH
2preparation:
Take Fullerene C20, graphene oxide GO, above-mentioned PTC-NH
2, grind to form fine-powdered, be transferred in PBS, after ultrasonic disperse, stir continuously under room temperature and spend the night, obtain C60-GO-PTC-NH
2;
(3) two anti-complex are prepared:
Get above-mentioned C60-GO-PTC-NH
2, add EDC/NHS, under room temperature, lucifuge stirs, and centrifuge washing, will precipitate resuspended to PBS, in above-mentioned solution, add detection antibody, and 4 DEG C of lucifuges stir, and use PBS centrifuge washing, precipitation be resuspended in PBS, 4 DEG C keep in Dark Place, and obtain described two anti-complex.
6. photoelectricity immunity biosensor according to claim 1, is characterized in that, described electrode is oxidation cigarette tin electrode.
7. the preparation method of photoelectricity immunity biosensor described in any one of claim 1-6, is characterized in that, comprise the following steps:
(1) graphene oxide GO is dripped: drip graphene oxide GO at electrode surface, make it be combined in electrode surface completely;
(2) active oxidation Graphene GO: utilize catalyzer, makes the carboxyl on graphene oxide GO activate completely;
(3) covalently bound capture antibody: the graphene oxide GO surface to above-mentioned activation drips capture antibody, and capture antibody is fully connected with graphene oxide GO;
(4) non-specific sites is closed: close the graphene oxide GO site obtained antibody and combine at large, form capture antibody layer;
(5) determined antigen in medium is caught: drip determined antigen to above-mentioned capture antibody layer, fully react, obtain determined antigen layer;
(6) detect antibody layer preparation: wash away above-mentioned unconjugated determined antigen, dry, drip and put forward described two anti-complex, carry out lucifuge, room temperature reaction, obtain detection antibody layer, carried out the amount of detectable antigens by the power change of two anti-complex photocurrents.
8. the preparation method of photoelectricity immunity biosensor according to claim 7, is characterized in that, comprise the following steps:
(1) drip graphene oxide GO: get graphene oxide GO, be dispersed in PBS, be added drop-wise to treated oxidation cigarette tin electrode surface, ambient temperatare is put, until it is combined in electrode surface completely;
(2) active oxidation Graphene GO: rinsed in PBS solution by the above-mentioned electrode adding GO and wash, wash away the GO not being combined in electrode surface, drips EDC/NHS, and room temperature is placed, and the carboxyl on GO is activated completely;
(3) covalently bound capture antibody: rinsed in PBS solution by step (2) the electrode obtained and wash, wash away the EDC/NHS do not utilized, dry, drips capture antibody, reacts, capture antibody is fully connected with GO at 4 DEG C;
(4) non-specific sites is closed: rinsed in PBS solution by step (3) the electrode obtained and wash, wash away unconjugated capture antibody, dry, drip bovine serum albumin(BSA), room temperature is placed, and closes the site obtained antibody and combine at large, forms capture antibody layer;
(5) catch the determined antigen in medium: rinsed in PBS solution by step (4) the electrode obtained and wash, wash away unnecessary BSA, dry, drip antigen, room temperature reaction, obtains determined antigen layer;
(6) antibody layer preparation is detected: rinsed in PBS solution by step (5) the electrode obtained and wash, wash away unconjugated antigen, dry, drip described two anti-complex, carry out lucifuge, room temperature reaction, obtains detection antibody layer, carrys out detectable antigens amount by the power change of two anti-complex photocurrents.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510734068.7A CN105424776A (en) | 2015-11-03 | 2015-11-03 | Biosensor based on carbon nano composite material and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510734068.7A CN105424776A (en) | 2015-11-03 | 2015-11-03 | Biosensor based on carbon nano composite material and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105424776A true CN105424776A (en) | 2016-03-23 |
Family
ID=55503121
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510734068.7A Pending CN105424776A (en) | 2015-11-03 | 2015-11-03 | Biosensor based on carbon nano composite material and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105424776A (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861295A (en) * | 2016-05-24 | 2016-08-17 | 长沙医学院 | Biosensor for detecting salmonella typhimurium and preparation and detection methods |
| CN105928997A (en) * | 2016-07-11 | 2016-09-07 | 山东理工大学 | Preparation method and application of immunosensor based on Au-GQD@PtPd |
| CN107037013A (en) * | 2017-04-21 | 2017-08-11 | 济南大学 | A kind of preparation and application of the optical electro-chemistry cell sensor of Sensitive Detection cancer cell |
| CN107422018A (en) * | 2017-06-29 | 2017-12-01 | 东南大学 | A kind of detection TNF α photoelectricity immunosensor and its preparation method and application |
| CN108896632A (en) * | 2018-04-11 | 2018-11-27 | 东南大学 | A kind of optical electro-chemistry immunosensor and the preparation method and application thereof measuring α-Fetoprotein |
| CN109580727A (en) * | 2018-12-14 | 2019-04-05 | 中国科学院上海微***与信息技术研究所 | A method of the detection organic amine escaping gas based on tetracarboxylic acid dianhydride |
| CN110702924A (en) * | 2019-11-08 | 2020-01-17 | 东南大学 | Photoelectrochemical immunosensor for detecting S100B and preparation method and application thereof |
| CN115219569A (en) * | 2022-06-22 | 2022-10-21 | 郑州大学 | Sensor for detecting tumor cells by artificial enzyme, and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013014538A2 (en) * | 2011-07-25 | 2013-01-31 | American University In Cairo | Single-domain antibodies and graphene coated magnetic metal nanoparticles conjugate and methods for using the same |
| KR20130106044A (en) * | 2012-03-19 | 2013-09-27 | 한국과학기술원 | Biosensor using the graphene oxide by the polymer of enzyme substrate and method of detecting using it |
-
2015
- 2015-11-03 CN CN201510734068.7A patent/CN105424776A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013014538A2 (en) * | 2011-07-25 | 2013-01-31 | American University In Cairo | Single-domain antibodies and graphene coated magnetic metal nanoparticles conjugate and methods for using the same |
| KR20130106044A (en) * | 2012-03-19 | 2013-09-27 | 한국과학기술원 | Biosensor using the graphene oxide by the polymer of enzyme substrate and method of detecting using it |
Non-Patent Citations (3)
| Title |
|---|
| CHENGGUO HU ET.AL: "Ultrasensitive All-Carbon Photoelectrochemical Bioprobes for Zeptomole Immunosensing of Tumor Markers by an Inexpensive Visible Laser Light", 《ANALYTICAL CHEMISTRY》 * |
| 刘中原: "基于纳米材料构建的新型电流型免疫传感器及电化学适体传感器研究", 《中国优秀硕士学位论文全文数据库（电子期刊）信息科技辑》 * |
| 惠俊敏: "新型电化学生物传感器的构建与应用", 《中国优秀硕士学位论文全文数据库（电子期刊）医药卫生科技辑》 * |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105861295A (en) * | 2016-05-24 | 2016-08-17 | 长沙医学院 | Biosensor for detecting salmonella typhimurium and preparation and detection methods |
| CN105928997A (en) * | 2016-07-11 | 2016-09-07 | 山东理工大学 | Preparation method and application of immunosensor based on Au-GQD@PtPd |
| CN107037013A (en) * | 2017-04-21 | 2017-08-11 | 济南大学 | A kind of preparation and application of the optical electro-chemistry cell sensor of Sensitive Detection cancer cell |
| CN107037013B (en) * | 2017-04-21 | 2019-07-05 | 济南大学 | A kind of preparation and application of the optical electro-chemistry cell sensor of Sensitive Detection cancer cell |
| CN107422018B (en) * | 2017-06-29 | 2019-06-18 | 东南大学 | A kind of photoelectricity immunosensor and its preparation method and application detecting TNF-α |
| CN107422018A (en) * | 2017-06-29 | 2017-12-01 | 东南大学 | A kind of detection TNF α photoelectricity immunosensor and its preparation method and application |
| CN108896632A (en) * | 2018-04-11 | 2018-11-27 | 东南大学 | A kind of optical electro-chemistry immunosensor and the preparation method and application thereof measuring α-Fetoprotein |
| CN108896632B (en) * | 2018-04-11 | 2021-01-05 | 东南大学 | Photoelectrochemical immunosensor for determining alpha fetoprotein content and preparation method and application thereof |
| CN109580727A (en) * | 2018-12-14 | 2019-04-05 | 中国科学院上海微***与信息技术研究所 | A method of the detection organic amine escaping gas based on tetracarboxylic acid dianhydride |
| CN109580727B (en) * | 2018-12-14 | 2021-05-14 | 中国科学院上海微***与信息技术研究所 | Method for detecting organic amine volatile gas based on perylene tetracarboxylic dianhydride |
| CN110702924A (en) * | 2019-11-08 | 2020-01-17 | 东南大学 | Photoelectrochemical immunosensor for detecting S100B and preparation method and application thereof |
| CN115219569A (en) * | 2022-06-22 | 2022-10-21 | 郑州大学 | Sensor for detecting tumor cells by artificial enzyme, and preparation method and application thereof |
| CN115219569B (en) * | 2022-06-22 | 2024-03-12 | 郑州大学 | Sensor for detecting tumor cells by artificial enzyme and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105424776A (en) | Biosensor based on carbon nano composite material and preparation method thereof | |
| Shen et al. | Recent development of sandwich assay based on the nanobiotechnologies for proteins, nucleic acids, small molecules, and ions | |
| Ma et al. | Electrochemiluminescent immunosensing of prostate-specific antigen based on silver nanoparticles-doped Pb (II) metal-organic framework | |
| Li et al. | Gold nanoparticle amplified optical microfiber evanescent wave absorption biosensor for cancer biomarker detection in serum | |
| Han et al. | A label-free electrochemical impedance cytosensor based on specific peptide-fused phage selected from landscape phage library | |
| Xu et al. | Disposable electrochemical immunosensor by using carbon sphere/gold nanoparticle composites as labels for signal amplification | |
| Aydın et al. | An impedimetric immunosensor for highly sensitive detection of IL-8 in human serum and saliva samples: A new surface modification method by 6-phosphonohexanoic acid for biosensing applications | |
| Liu et al. | SPR quantitative analysis of direct detection of atrazine traces on Au-nanoparticles: nanoparticles size effect | |
| Zheng et al. | Competitive near-infrared PEC immunosorbent assay for monitoring okadaic acid based on a disposable flower-like WO3-Modified screen-printed electrode | |
| CN103608675B (en) | Luminescent polymer cyclic amplification | |
| CN102636649B (en) | Kit for detecting carcinoembryonic antigen based on antibody functionalized magnetic nanometer material and up-conversion fluorescence nanometer material | |
| Kim et al. | High-performance biosensor using a sandwich assay via antibody-conjugated gold nanoparticles and fiber-optic localized surface plasmon resonance | |
| CN103163076B (en) | Method for detecting L-cysteine concentration by applying of circular dichroism | |
| Xiang et al. | Novel ultralow-potential electrochemiluminescence aptasensor for the highly sensitive detection of zearalenone using a resonance energy transfer system | |
| Wang et al. | Chemiluminescence excited photoelectrochemical competitive immunosensing lab-on-paper device using an integrated paper supercapacitor for signal amplication | |
| Özyurt et al. | Biosensing strategies for diagnosis of prostate specific antigen | |
| Nie et al. | A highly sensitive capillary-based immunosensor by combining with peroxidase nanocomplex-mediated signal amplification for detection of procalcitonin in human serum | |
| Liang et al. | A novel, label-free immunosensor for the detection of α-fetoprotein using functionalised gold nanoparticles | |
| Chen et al. | Nanobody-based label-free photoelectrochemical immunoassay for highly sensitive detection of SARS-CoV-2 spike protein | |
| Lenyk et al. | Dual‐Transducer Malaria Aptasensor Combining Electrochemical Impedance and Surface Plasmon Polariton Detection on Gold Nanohole Arrays | |
| Abd Manaf et al. | Recent advancement in sensitive detection of carcinoembryonic antigen using nanomaterials based immunosensors | |
| Chang et al. | Facile engineered polymeric microdevice via co-coupling of phenylboronic acid and Protein A for oriented antibody immobilization enables substantial signal enhancement for an enhanced fluorescence immunoassay | |
| Chen et al. | Near-infrared surface plasmon resonance sensor with a graphene-gold surface architecture for ultra-sensitive biodetection | |
| Wang et al. | Fabrication of a versatile aptasensing chip for aflatoxin B1 in photothermal and electrochemical dual modes | |
| Guo et al. | Dual-recognition immune-co-chemical ECL-sensor based on Ti, Mg@ N-CDs-induced and novel signal-sensing units Poly (DVB-co-PBA)-reported for alpha-fetoprotein detection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160323 |